The long-term goal of this project is to elucidate basic mechanisms underlying sex determination (the decision to develop testes or ovaries) and gonadal differentiation. Such information provides the foundation for understanding and ultimately preventing defects in gonad development and function. The specific aims outlined here harness the power of comparative and functional genomics to identify an evolutionary conserved set of sex-determining genes in amniotic vertebrates, including humans. In Specific Aim 1, we will clone & sequence genes that are differentially expressed between incipient testis and ovary in alligator and mouse embryos. We will then confirm sexually dimorphic expression of these genes. Finally, we will compare cDNA sequences to identify novel genes that are conserved in sequence between these species and query electronic databases to identify homologous genes in humans. In Specific Aim 2, we will use fluorescence in situ hybridization to determine which gonadal cell types express the conserved sex-determining genes (Sertoli, Leydig, Granulosa, or Thecal cells, as well as primordial germ cells). These studies pioneer the fusion of comparative genomics and functional genomics to identify coexpressed gene orthologs in a massively parallel manner. Using this approach, we expect to identify novel genes that are conserved in sequence, sexually dimorphic expression, and cell-type specific expression in vertebrates with distinct modes of sex determination. Such genes are highly likely to have the same function in sex determination in all vertebrates, including humans. This approach promises to speed the rate of discovery of sex-determining genes in humans, which to date has been slow and haphazard.